Transforming growth factor-beta (TGF-beta) is a potent inhibitor of human mammary epithelial proliferation. During mammary carcinogenesis most breast cancer cells become resistant to the growth inhibitory effects of TGF-beta; TGF-beta then promotes breast cancer tumor formation. Fundamental gaps in knowledge exist in terms of the mechanisms (1) by which breast cancers become TGF-beta resistant and (2) by which TGF-beta functions as both a tumor suppressor and a tumor promoter during mammary carcinogenesis. The type III TGF-beta receptor (TbetaRIII) has an emerging yet poorly understood role in regulating TGF-beta signaling and mammary carcinogenesis. Based on preliminary studies establishing the scaffolding protein beta-arrestin2 as a TbetaRlII-interacting protein, the following hypothesis is proposed: Interaction of TbetaRIII with beta-arrestin2 during mammmary carcinogenesis mediates (1) the internalization and downregulation of TGF-beta receptors, resulting in resistance to TGF-beta-mediated inhibition of proliferation and (2) stimulation of TbetaRIII-dependent signaling to MAP kinase pathways promoting tumor formation. This hypothesis will be addressed by four Specific Aims. Specific Aim 1: The TbetaRIII/beta-arrestin2 interaction will be characterized and the effects of TbetaRIII phosphorylation and TbetaRIII-interacting proteins (GIPC, TbetaRII) on the TbetaRIII/beta arrestin2 interaction and beta-arrestin2-mediated TbetaRIII internalization in human breast cancer cells will be established. Specific Aim 2: The effect of beta-arrestin2 on TbetaRIII expression, downregulation, TGF-beta signaling and TGF-beta-mediated inhibition of proliferation in human breast cancer cells will be established to define whether TbetaRIII/beta-arrestin2 mediates the tumor suppressor effects of TGF-beta during mammary carcinogenesis. Specific Aim 3: The effect of beta-arrestin2 on TbetaRIII-dependent signaling to MAP kinase pathways and on the proliferation, motility, survival and anchorage-independent growth of human breast cancer cells will be established to define whether TbetaRIIl/beta-arrestin2 mediates the tumor promoting effects of TGF-beta during mammary carcinogenesis. Specific Aim 4: The effect of altering beta-arrestin2 or GIPC expression or the ability of TbetaRIII to interact with beta-arrestin2 or GIPC on the balance of TbetaRIII-mediated Smad-dependent and Smad-independent signaling and on the tumorigenicity/metastatic potential of breast cancer cells in vivo will be established to determine whether TbetaRIII through beta-arrestin2 regulates TGF-beta signaling during mammary carcinogenesis. These studies will define mechanisms for TGF-beta-resistance, for Smad-independent signaling and for the dichotomous role of TGF-beta in mammary carcinogenesis and aid in targeting the TGF-beta signaling pathway for the chemoprevention and treatment of human breast cancers and other human cancers in which TGF-beta has a defined role.